Microtubule (MT) polymerization has been extensivly studied in vitro, but numerous questions about MT behavior in vivo remain unaddressed. Tools are now available to rectify this situation. We will make neurotubulin labeled with dichlorotriazinyl amino fluorescein and introduce it into living cells by microinjection. We will follow its behavior in vivo with a low light-level TV system coupled to an image processing computer. We hope to learn where MT subunit incorporate in vivo by following the fluorescence in cells immediately after injection. Immunocytochemistry at the EM level using antibodies to fluorescein will provide related information at high space resolution. Fluorescence incorporation behavior should also tell us whether MTs "treadmill" in vivo. In a second experimental strategy, we will inject cells as above, let the fluorescence equilibrate and then follow subunit turnover by measuring the fluorescence redistribution after photobleaching (FRAP). Studies of interphase cells by FRAP will provide additional evidence about MT treadmilling. Studies of mitotic cells by FRAP should allow us to learn the motion of chromosomes relative to their fibers during anaphase and determine the modes of subunit addition to and loss from mitotic MTs. We should also learn whether interzone MTs slide in anaphase. We will study the motions of interphase MTs relative to their sites of initiation as they polymerize and depolymerize in response to both natural and experimental stimulation. We will then take advantage of existing information about MTs and MT associated proteins to try to identify the factors that regulate MT polymerization and/or treadmilling in vivo. To this end we will manipulate the concentrations and post-translational modifications of MT associated proteins and of tubulin itself. The role of Ca-calmodulin will be explored. Suggestive results from experiments in vivo will be used to design polymerization experiments in vitro to analyze regulatory phenomena in a more specifically defined environment.